1. Field of the Invention
The invention relates to the field of genetic breeding for aquatic animals, in particularly to a recombinant gene which enhances the hypoxia tolerance. The invention also relates to the use of such recombinant gene for genetically improving farmed species.
2. Description of the Related Art
Dissolved oxygen (DO) is the most important environmental factor in aquiculture, which may be affected by temperature, diurnal rhythm, seasonal variation and eutrophication, etc. Fish are very sensitive to the variation of DO condition in the water. Low DO reduces the growth rate of fish, induces endocrine disorders of fish, impacts metabolism and reproduction features of fish, changes the behavior and distribution of fish, and even results in the death of fish. Therefore, the abundance, diversity and capture amount of fish are all severely impaired in the water areas affected by low DO. As a result, it is practically worth to develop novel farmed species with the hypoxia tolerance using transgenic technology.
Currently, the study of improving hypoxia tolerance in fish using transgenic technology is still a presumption. The hypoxia tolerance in fish mainly relies on the contents and types of globulin families and oxygen affinity (Giles M. A (1991). Strain differences in hemoglobin polymorphism, oxygen consumption, and blood oxygen equilibria in three hatchery broodstocks of Arctic charr, Salvelinus alpinus. Fish Physiology and Biochemistry 9: 291-301. 2, Terwilliger N. B (1998). Functional adaptations of oxygen-transport proteins. J. Exp. Biol. 201(8): 1085-1098. 3, Skjæaasen J. E., Nilsen T., Meager J. J., Herbert N. A., Moberg O., Tronci V., Johansen T., Salvanes A. G. V (2008). Hypoxic avoidance behaviour in cod (Gadus morhua L.): The effect of temperature and haemoglobin genotype. Journal of Experimental Marine Biology and Ecology; 358; 70-77.). Four types of globulins were found in human and other vertebrates: haemoglobin (Hb), myoglobin (Mb), neuroglobin and cytoglobin. These four types of globulins vary to some extent in structure, tissue distribution and function (4, Pesce A., Bolognesi M., Bocedi A., Ascenzi P., Dewilde S., Moens L., Hankeln T., Burmester T. (2002). Neuroglobin and cytoglobin. Fresh blood for the vertebrate globin family. EMBO Rep. 3(12):1146-1151.). In mammals, haemoglobin (Hb) and myoglobin (Mb) may be induced by low oxygen and hypoxia (5, Hoppeler H., Vogt M. (2001). Muscle tissue adaptations to hypoxia. J. Exp. Biol. 204: 3133-3139. 6, Vogt M., Puntschart A., Geiser J., Zuleger C., Billeter R., and Hoppeler H. (2001). Molecular adaptations in human skeletal muscle to endurance training under simulated hypoxic conditions. J. Appl. Physiol. 91: 173-182. 7, Nitta T., Xundi X., Hatano E., Yamamoto N., Uehara T., Yoshida M., Harada N., Honda K., Tanaka A. & Sosnowski D. (2003). Myoglobin gene expression attenuates hepatic ischemia reperfusion injury. J. Surg. Res. 110: 322-331.).
Strictly speaking, Vitreoscilla stercoraria is an aerobe, but they are able to adapt to hypoxia by expressing soluble haemoglobin (Vitreoscilla hemoglobin, VHb) which consists of 2 identical subunits of 15,775 Da and 2 heme molecules (Wakabayashi S., Matsubara H., Webster D. A. (1986). Primary sequence of a dimeric bacterial hemoglobin from Vitreoscilla. Nature 322:481-483.). Although VHb does not increase intracellular oxygen concentration, it can accelerate oxygen delivery in a low oxygen condition due to the high dissociation rate constant between VHb and oxygen molecules, and thus improves respiration and energy metabolism. Therefore, hypoxia tolerance in fish may be prospectively enhanced by introducing VHb gene into fish, so as to develop novel farmed species with hypoxia tolerance.
Zebrafish (Danio rerio) is a fish model currently widely used in many investigations such as Developmental Biology and Hydrobiont Technology (Westerfield M. (1993). The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio). University of Oregon Press, Eugene, Oreg.). A new method, where zebrafish is used as a model to study the improvement of the ability of recipient fish to tolerate low oxygen stress, results in an instructive significance for breeding novel economically farmed species with tolerance to low DO stress. Based on breeding of the transgenic zebrafish with the hypoxia tolerance, the recombination technique is further applied to other important economically farmed species such as blunt snout bream (Megalobrama amblycephala) and common carp (Cyprinus carpio L.). The resultant vhb transgenic blunt snout bream and common carp also have the hypoxia tolerance.